When phosphatidylinositol 4,5-bisphosphate is hydrolyzed through the activation of a receptor on a cell membrane, inositol 1,4,5-trisphosphate (IP3), which is an intracellular second messenger, is generated. IP3 binds to an IP3 receptor (IP3R), thereby inducing Ca2+ release from organelles for intracellular calcium storage (mainly, the endoplasmic reticulum). In this IP3/Ca2+ signaling pathway, the IP3 receptor plays a role in converting the IP3 signal into Ca2+ signal (M. J. Berridge, Nature (1993) 361: 315-325; M. J. Berridge et al., Nat. Rev. Mol. Cell. Biol. (2000) 1: 11-21; T. Furuichi and K. Mikoshiba, J. Neurochem. (1995) 64: 953-960).
The IP3 receptor is a tetrameric intracellular IP3-gated Ca2+ release channel. In mammals, there exist 3 different types of IP3 receptors (i.e., type 1, type 2, and type 3) (T. Furuichi et al., Nature (1989) 342: 32-38; T. Sudhof et al., EMBO J. (1991) 10: 3199-3206; 0. Blondel et al., J. Biol. Chem. (1993) 268: 11356-11363). Of them, the type 1 IP3 receptor (IP3R1) is expressed at high levels in the central nervous system and particularly in the cerebellum (P. F. Worley et al., Nature (1987) 325: 159-161; T. Furuichi et al., Recept. Channels (1993) 1: 11-24). Mouse IP3R1 comprises 2749 amino acids and has 3 functionally different regions. Specifically, an IP3-binding domain is present in the vicinity of the N-terminus, a channel-forming domain having a six-transmembrane region is present in the vicinity of the C terminus, and a control region is present between the two regions. The deletion mutant analysis of the IP3-binding domain revealed that the amino acids 226-578 of the IP3 receptor was a minimum region required for specific and high-affinity binding of a ligand. This region is referred to as the IP3 binding core.
With an increase of cytoplasmic Ca2+ concentration by activation of the IP3 receptor, the activities of a wide variety of downstream target molecules are controlled. These downstream target molecules play important roles in wide-ranging cellular responses including fertilization, development, proliferation, secretion, synaptic plasticity, and the like.
The present inventors have previously discovered a novel IP3 receptor-binding protein and named it “IRBIT” (IP3R-binding protein released with inositol 1,4,5-trisphosphate) (JP2004-129612A). The IP3 receptor is widely distributed in various tissues and cells of mammals such as humans and mice (e.g., in the brain, heart, liver, kidney, pancreas, and thymus gland). Accordingly, IRBIT is inferred to be present also in such tissues or cells. The amino acid and nucleotide sequences of mouse IRBIT have been determined by the present inventors (JP2004-129612A, H. Ando et al., J. Biol. Chem. (2003) 278: 10602-10612). Such IRBIT comprises 530 amino acids. Human IRBIT and mouse IRBIT share 100% identity. The region for binding to the IP3 receptor is present in the N-terminal region of IRBIT, corresponding to amino acids 1-104 in a human or a mouse.
IRBIT is characterized in that: (1) IRBIT is a neutral protein (presumed pI: 6.48) in which the N-terminal region is relatively acidic (presumed pI: 4.98); (2) a plurality of phosphorylation sites are localized in a concentrated manner in the N-terminal region, so that phosphorylation is predicted to be necessary for interaction with IP3R1; (3) the lysine residue at position 508, which is essential for the binding of IP3R1 to IP3, is also essential for interaction with IRBIT; (4) IRBIT is dissociated by IP3 from interaction with IP3R1; and (5) because IRBIT is dissociated from IP3R1 and is extracted from crude microsome fractions by high salt, it is inferred that its interaction with IP3R1 takes place due to electrostatic binding, for example (JP2004-129612A).
IRBIT has the property that it binds to the IP3 binding region of the IP3 receptor and is dissociated in vitro from the IP3 receptor by IP3. Therefore, it has also been revealed that IRBIT has a function of suppressing the activity of the IP3 receptor by suppressing the binding of IP3 to the IP3 receptor (JP2004-129612A).
The present inventors have now found target molecules of IRBIT and important biological in vivo functions of IRBT as a tertiary messenger, as described below.